DNA polymerases achieve accurate DNA replication through a delicate balance between primer elongation and proofreading. While insufficient proofreading results in DNA replication errors, indiscriminate removal of correct along with incorrect nucleotides is wasteful and may prevent completion of DNA synthesis. The transition between polymerization and proofreading modes is proposed to be governed by a kinetic barrier that prevents proofreading unless the rate of primer elongation is significantly reduced by the presence of an incorrect base pair at the primer-terminus. We have used mutational analysis, coupled with a sensitive, fluorescence-based assay to characterize intermediate steps in the proofreading pathway. A highly fluorescent complex forms between the bacteriophage T4 DNA polymerase and DNA primer-templates labeled at the 3' terminus with the base analog 2-aminopurine. Formation of the fluorescent complex appears to be a rate-determining step in the proofreading pathway and is impaired for several mutator T4 DNA polymerases with amino acid substitutions in the exonuclease domain. Although these mutant DNA polymerases are proficient in hydrolysis, their reduced ability to form the fluorescent complex imposes a higher kinetic barrier. As a consequence, the mutant DNA polymerases proofread less frequently, resulting in more DNA replication errors.